Calcium restriction induces cardenolide accumulation in cell suspension cultures of Digitalis thapsi L.

Summary The removal of calcium ions from Murashige and Skoog culture medium induced a marked increase in the accumulation of cardenolides in cell suspension cultures of Digitalis thapsi. Cell viability was not affected although growth was slightly reduced. Strontium ions could substitute for calcium in inhibiting cardenolide production, this effect of calcium being reversed by the addition of LaCl₃ or ethyleneglycol-bis-(-aminoethyl ether)-N,N-tetraacetic acid. The results suggest that calcium, apart from its general effects on growth, may play a role in the regulation of cardenolide metabolism in a concentration dependent manner.Abbreviations BA 6-benzylaminopurine - 2 4-D 2,4-dichlorophenoxyacetic acid - EGTA ethyleneglycol-bis-(-aminoethyl ether)-N,N-tetraacetic acid - FW fresh weight - MS Murashige and Skoog (1962)     

小剂量洋地黄对急性心肌梗死PCI术后合并心力衰竭患者心率变异性的影响

目的:探讨早期应用小剂量洋地黄类药物对急性心肌梗死(Acute myocardial infarction,AMI)行经皮冠状动脉介入治疗(Percutaneous coronary intervention,PCI)术后合并心力衰竭患者心率变异性(Heart rate variability,HRV)的影响。方法:入选32例在发病24小时内接受PCI治疗且合并心力衰竭的AMI患者,再灌注后随机分为洋地黄组(西地兰0.2 mg,n=17)和对照组(生理盐水20 m L,n=15)。在用药前、用药后30分钟、用药后3小时、用药后6小时、用药后12小时、用药后24小时进行5分钟HRV分析。结果:1洋地黄组的心率在用药6小时后显著小于对照组(P0.05);2洋地黄组SDNN在用药后3小时-6小时显著大于对照组(P0.05),两组RMSSD比较无显著统计学差别(P0.05);3洋地黄组LFnorm在用药后3小时-6小时显著大于对照组(P0.05);用药3小时后,洋地黄组HFnorm显著大于对照组(P0.05),LF/HF显著小于对照组(P0.05)。结论:小剂量洋地黄可以显著降低AMI PCI术后合并心力衰竭患者的心率、逆转迷走神经与交感神经活性的失衡状态,改善HRV。     

Incorporation of 5α-furostan-3β,26-diol into tigogenin by digitalis lanata

2,2′,4,4′-³H₄-dihydrotigogenin was converted by Digitalis lanata plants into tigogenin.     

'Cholesterol side-chain cleaving enzyme' aktivität in keimlingen und in vitro kultivierten geweben von Digitalis purpurea

Experiments with cholesterol-26-¹⁴C gave evidence for the occurrence of cholesterol side-chain cleaving enzyme in seedlings of Digitalis purpurea. Tissue cultures of D. pupurea failed to show cholesterol side-chain cleaving activity, which may be the reason why tissue cultures are unable to biosynthesize cardenolide glycosides.     

The dissipation of excess excitation energy in British plant species

The reversible dissipation of excitation energy in higher plants is believed to protect against light-induced damage to the photosynthetic apparatus. This dissipation is measured as the non-photochemical quenching of chlorophyll fluorescence. A method is described whereby the saturated capacity for rapidly reversible non-photochemical quenching can be compared between plant species. This method was applied to 22 common British plant species whose habitat was quantified using an index that describes shade tolerance. An association was found between occurrence in open habitats and a high capacity for non-photochemical quenching. It was found that, whilst this capacity was species dependent, it did not depend upon the conditions under which the plant was grown. The possible role of zeaxanthin as a determinant of quenching capacity was examined by measuring the contents of xanthophyll cycle carotenoids for each species. Comparing species, no correlation was seen between the saturated level of non-photochemical quenching and zeaxanthin content expressed relative to either total carotenoid or to chlorophyll. When zeaxanthin was expressed relative to the amount of xanthophyll cycle intermediates (zeaxanthin, antheraxanthin and violaxanthin), a weak correlation was seen.     

Purification and characterisation of the cardenolide-specificβ-glucohydrolase CGH II from Digitalis lanata leaves

A cardenolide-hydrolysing β-D-glucosidase was isolated from young leaves of Digitalis lanata. Since this enzyme differs from the cardenolide glucohydrolase (CGH) described and characterised previously, it was termed cardenolide glucohydrolase II (CGH II). CGH II was detected in various Digitalis tissue cultures as well as in young leaves of D. lanata. The latter source was used as the starting material for the isolation and purification of CGH II. The specific enzyme activity reached about 15 pkat·mg^–1 protein in buffered leaf extracts. Optimal CGH II activity was seen at around pH 6.0 and 50 °C. CGH II was purified about 600-fold by anion exchange chromatography, size exclusion chromatography and hydroxyapatite chromatography. The apparent molecular mass of CGH II was 65 kDa as determined by SDS-PAGE. CGH II exhibited a high substrate specificity towards cardenolide disaccharides, especially to those with a 1-4-β-linked glucose-digitoxose moiety such as glucoevatromonoside. The K_m- and V_max-values for this particular substrate were calculated to be 101 μM and 19.8 nkat·mg^–1 protein, respectively.     

Kinetic model for biotransformation of digitoxin in plant cell suspension culture ofDigitalis lanata

Batch suspension cultures ofDigitalis lanata plant cell were performed to investigate the biotransformation of digitoxin.Digitalis lanata K3OHD plant cells were used to biotransform digitoxin into deacetyllanatoside C. A kinetic model was proposed to describe cell growth, substrate consumption, depletion of digitoxin, formation and depletion of digoxin and purpureaglycoside A, and formation of deacetyllanatoside C. The digoxin and purpureaglycoside A are intermediates of deacetyllanatoside C formation from digitoxin. Interactions between extracellular and intracellular compounds were considered. The proposed model could accurately predict cell growth, substrate consumption and product synthesis. And it can provide a useful framework for quantitative analysis of biotransformation in a plant cell culture system.     

Pollen plant formation from anther cultures of Digitalis obscura L.

Factors favouring pollen callus proliferation, induction of embryogenesis and plant regeneration from cultured anthers of Digitalis obscura L. were determined. The presence of auxins was essential for cell proliferation and morphogenesis, and incubation in darkness singificantlyincreased these responses. Callus proliferation usually preceded embryo development, although sometimes direct embryogenesis was observed. On the other hand, bud differentiation was achieved only when callus was transferred to media containing cytokinin or several auxin/cytokinin combinations. Different ploidy levels] were observed in the regenerated plants, with approximately 50% being haploid.     

毛地黄(Digitalis purpurea)叶离体培养过程中光质与培养基对器官发生的交互作用

培养基成分影响毛地黄叶外值体的生长,不含BA的培养基中芽不能发生,无NAA的培养基中无根发生。光质的效应与培养基成分有关,黄、蓝、绿光在未加有机成分(NAA为0.ling·L~(-1))的培养基中能促进芽的生长,当NAA为0.5 mg·L~(-1)时则抑制芽的生长。红光、黑暗处理与培养基成分关系不大,一般均抑制发芽;根的发生不需要光。光质和培养基之间有交互作用。     

Identification and Quantification of Several Mammalian Steroid Hormones in Plants by UPLC-MS/MS

We have developed an effective method for the isolation, identification, and quantification of several mammalian steroid hormones and their metabolites in different plant tissues. The purification protocol was based on solid-phase extraction (SPE) combined with immunoaffinity chromatography (IAC) using immobilized generic polyclonal anti-Δ⁴-3-keto-steroid antibodies covalently bound to Affi-Gel 10 sorbent. The antibodies were characterized by means of enzyme-linked immunosorbent assay (ELISA). The detection limit of the ELISA was 6.0 × 10⁻¹⁰ mol L⁻¹ and cross-reactivity with most Δ⁴-3-keto-steroids was very high as predicted (68–122%). The IAC allowed fast, single-step purification of different plant extracts prior to analysis by ultra-performance liquid chromatography-electrospray tandem mass spectrometry [UPLC-ESI(+)-MS/MS]. In multiple-reaction-monitoring (MRM) mode, the detection limit of the method for most of the steroids analyzed was close to 10 fmol and the response was linear up to 50 pmol injected. The analytical accuracy was validated using tobacco leaf samples spiked with known amounts of authentic and deuterium-labeled standards. The newly developed method was capable of detecting and quantifying at least 12 specified steroid compounds in plant extracts. In the analyzed extracts from three plant species, that is, common foxglove (Digitalis purpurea L.), tobacco (Nicotiana tabacum L.), and elecampane inula (Inula helenium L.), four endogenous steroids were detected, identified, and quantified. Progesterone was found in all three plants at concentrations comparable to those reported in previous studies. Three other steroids, androstendione, 17α-hydroxyprogesterone, and 16-dehydroprogesterone, were identified for the first time in plant extracts. 17α-Hydroxyprogesterone and 16-dehydroprogesterone occurred at significant concentrations in D. purpurea, whereas androstendione was found in N. tabacum and I. helenium but not in D. purpurea.